Although the development of recombinant DNA technology and the identification and isolation of proteins mediating a wide variety of biological activities has enabled the development of new drug therapies, proteins in general suffer from the disadvantage of susceptibility to breakdown by digestive and other enzymes. This means not only that these agents usually have to be administered by injection, but that they also have a short half-life in the body.
The biological activities of a protein rely on the three-dimensional structure of the protein molecule, which results predominantly from a balance between a variety of different non-covalent interactions. In an attempt to improve the stability and acceptability of protein pharmaceuticals, both relatively short peptide sequences encompassing the active site of the protein and synthetic molecules which adopt a three-dimensional structure resembling the active site have been extensively investigated. Structurally-constrained peptides in which a framework is maintained by disulphide bonds as well as by non-covalent interactions, and cyclic peptide or peptidomimetic systems in which the cyclisation provides the structural constraint, provide two particularly attractive approaches to stabilisation of these molecules.
Cyclic peptides show a wide variety of potent biological activities. They have been extensively explored in the drug development process as a means of introducing conformational constraints for the evaluation of the structural, conformational and dynamic properties that are critical to biological activity. Some cyclic peptides are useful as drugs in their own right. Others have been engineered to provide a multitude of functions, including novel biological properties, platforms for the development of protein mimetics, nanotechnology, specific metal coordination sites, and catalysts, to name a few.
Cyclisation may be accomplished by disulfide bond formation between two side chain functional groups, amide or ester bond formation between one side chain functional group and the backbone α-amino or carboxyl function, amide or ester bond formation between two side chain functional groups, or amide bond formation between the backbone α-amino and carboxyl functions.
The potential utility of this class of compound in any application is hindered by difficulties in synthesising the compounds. Whilst the synthesis of the linear precursors generally proceeds in high yield and purity, the final cyclisation reaction can be troublesome, resulting in low yields and/or impure products. This is particularly so for cyclic peptides of fewer than seven amino acid residues, with synthesis of cyclic tetrapeptides resulting in little or no cyclic material.
These cyclisation reactions have been traditionally carried out at high dilution in solution. With the advent of orthogonal protection strategies and new resins for solid phase peptide synthesis, cyclisation has been accomplished while the peptide is attached to the resin. One of the most common ways of synthesising cyclic peptides on a solid support is by attaching the side chain of an amino acid to the resin. Using appropriate orthogonal protection strategies, the C- and N-termini can be selectively deprotected and cyclised on the resin after chain assembly. This strategy is widely used, and is compatible with either tert-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) protocols. However, it is restricted to peptides that contain appropriate side chain functionality to attach to the solid support. It is therefore not amenable to the combinatorial synthesis of arrays of cyclic peptides.
A number of approaches have been used in an attempt to achieve efficient synthesis of cyclic peptides.
Linkers
a) Activated Linkers
One procedure for synthesising cyclic peptides is based on cyclisation with simultaneous cleavage from the resin. After an appropriate peptide sequence is assembled by solid phase synthesis on the resin or a linear sequence is appended to resin, the deprotected amino group can react mildly with its anchoring active linkage to produce protected cyclic peptides, as shown schematically in Scheme 1.
Solid Phase Cyclic Peptide Synthesis with Activated Linkers
Various linkers that have been used for the synthesis of cyclic peptides, or are amenable to their synthesis, are shown in Table 1.
TABLE 1Examples of Activated Linkers Amendableto Solid Phase Cyclic Peptide SynthesisLinkerReferenceFridkin et al, 1965; Fridkin et al, 1968 Osapay and Taylor, 1990; Osapay et al, 1990 Rivaille et ali, 1980 Richter et al, 1994 Fridkin et al, 1972; Laufer et al, 1968.R = Peptide,  = support
These cleavage-by-cyclisation strategies produce protected cyclic peptides, necessitating a final deprotection step to synthesise the target cyclic material. The cyclisation reaction is generally slow and low in yield, because extended conformational preference of the linear analogue impedes the final cyclisation reaction.
b) Safety Catch Linkers
Extensions of these concepts include supports that can be selectively modified at the end of the assembly to increase the lability of the linker. These linkers are stable during peptide assembly, and are selectively activated, leading to cyclisation and cleavage from the resin. In general, a final deprotection step is required to yield the target cyclic peptide. Examples of linkers that can be used for this approach are shown in Table 2.
TABLE 2Examples of Safety Catch Linkers for Solid Phase Peptide SynthesisSafety CatchReagentActivated LinkerRef.H2O2Flanigan and Marshall, 1970 mcPBA/ DioxaneMarshall and Liener, 1970 H2O2Flanigan, 1971 HBrFlanigan, 1971 CH2N2Kenner et al, 1971 ICH2CNBackes et al, 1996 CH2N2Backes and Eliman, 1994
These strategies are again limited by the conformational preferences of the linear precursor.
c) Backbone Linkers
A simple extension of the concept of attaching the side chain to resin to achieve C— to N-cyclisation is the attachment of the backbone N to resin. Recently Jensen et al (1996) reported a backbone linker that has been used for synthesising linear peptides, diketopiperazines, peptide aldehydes and cyclic peptides (Jensen et al, 1998). There are several limitations to this process, these include difficulties in acylating the secondary amine to form the ‘linked’ amide bond and the fact that standard Fmoc SPPS leads to almost complete diketopiperazine formation at the dipeptide stage. Special protection strategies need to be employed to avoid this problem.

Backbone Linkers for Solid Phase Peptide Synthesis
Intraresin Chain Transfer
Another approach for synthesising cyclic peptides involves the attachment of a linker that contains two peptide attachment points to the resin, one of which is temporarily masked. Using standard solid phase techniques, the linear precursor is assembled on resin. The X and Y functionalities (Scheme 3) are then selectively unmasked and cyclised. Cleavage at the linker liberates the free C-terminal carboxylic acid group while the peptide is still attached to the resin. C- and N-cyclisation is then achieved by standard activation conditions, yielding cyclic peptides.

Linker Combination for Solid Phase Peptide Synthesis
This method is somewhat limited by the incorporation of the appropriate functionality X into a peptide sequence, and the complex deprotection strategies required. Once again, due to the extended nature of the linear precursors, cyclisation yields would be low.
Preorganising Peptides for Cyclisation
a) Reversible N-Substitution
The formation of a peptide ring, like any other cyclisation reaction, requires the generation of mutually reactive chain ends, and the reaction of these ends under conditions favouring intramolecular processes. The ease of formation of the ring is related to the conformational stability of the ring and to the losses of internal degrees of freedom that occur upon ring formation. Consequently the presence of turn-inducing amino acids such as Gly, Pro or a D-amino acid enhances the conformational stability of the ring and improves cyclisation yields. For linear peptides that do not contain amino acid residues that stabilise turn structures, the cyclisation reaction is likely to be an inherently improbable or slow process, due to the preference for extended conformations resulting in large strain upon ring formation.
This has led to the utilisation of various reversible chemical modifications of the peptide main chain, to enhance the cis amide bond conformation and hence reduce ring strain upon cyclisation, and to improve cyclisation yields. In the synthesis of cyclo-[Phe Phe Phe Phe] (SEQ ID NO:61), each amide N was substituted with a Boc (Cavelier-Frontin et al, 1993). In this instance the cyclisation yield increased from less than 1% to 27%. Similarly, the use of the N-(2-hydroxy-4-methoxybenzyl) (HMB) group as a reversible N-substituent has resulted in similar increases in yields of cyclic peptides (Ehrlich et al, 1996; Ehrlich et al, 1996), although no systematic study has been undertaken to quantify these effects. From the point of view of constructing peptide libraries it is impracticable to substitute every amide N of the linear precursor.
b) Ring Contraction
Ring contraction chemistry can be used for initial formation of larger flexible rings where the desired C- and N-termini are appropriately positioned to “snap shut” in a ring contraction reaction to yield the target cyclic peptide after deprotection. Ring contraction for the synthesis of cyclic peptides by intramolecular thiazolidine formation from linear unprotected peptide precursors (Scheme 4) has recently been reported (Botti et al, 1996). This procedure has the disadvantage of incorporation of the thiazolidine ring, and an additional stereo centre, into every sequence, and is not a generic procedure suitable for a combinatorial library approach.
Ring Contraction Chemistry for Synthesis of Cyclic Peptides
Several other research groups have also utilised ring contraction approaches for the synthesis of cyclic peptides (Camamero and Muir, 1997; Shao et al, 1998). These procedures either require the presence of a Cys or are restricted to cyclisation of peptides containing Gly at one of the termini, and are therefore not suitable for library development.
There is therefore a great need in the art for a mild, efficient, versatile synthetic strategy for the synthesis of cyclic peptides. We have now found that by introducing substituents or other moieties which preorganise peptides for cyclisation, cyclic peptides can be efficiently synthesized under mild conditions both in solution and on resin. These moieties, which we have termed peptide cyclisation auxiliaries, result in increased yields and purity of cyclic peptides. We have examined two approaches:                1. Positioning reversible N-amide substituents in the sequence.        2. Applying native ligation chemistry in an intramolecular sense.We have evaluated these for their improvements in the solution and solid phase synthesis of small cyclic peptides.        
We have systematically investigated the effects of preorganising peptides prior to cyclisation, and have developed new linkers to aid cyclic peptide synthesis. We have found surprising improvements in both yields and purity of products compared to the prior art methods. The combination of these technologies provides a powerful generic approach for the solution and solid phase synthesis of small cyclic peptides.
We have also developed linkers, and peptide cyclisation auxiliaries to aid cyclic peptide synthesis.
The ring contraction and N-amide substitution technology of the invention used in conjunction with the activated, safety catch, and backbone linker strategies of the invention provide improved methods for the solid-phase synthesis of cyclic peptides.